Since 23% of children born with HIV are born in Nigeria, our collaborators have made prevention of mother to child HIV transmission one of their highest program priorities. It is essential for viremic pregnant women to maintain durable viral suppression, so they do not transmit HIV to their unborn children. A major impediment to this is the presence of drug resistance mutations that evade the HIV antiretroviral treatments resulting in continued viremia. Currently at the Amino Kano Teaching Hospital antenatal care clinic viral load testing takes 2-4 weeks to receive results, and it takes an additional 4-6 weeks to receive sequencing results from the national laboratory. This turnaround time significantly increases the duration of viral exposure to the unborn child. We hypothesize that by providing a PCR-based test for drug resistance mutations, we will expedite determination of the most effective drug treatment regimens and reduce mother to child HIV transmission. In this R21, we propose to develop and test reagents and magnetic bead sample processing for detecting drug resistance mutations. We propose to achieve this by converting highly variable HIV sequences surrounding a known drug resistance mutation into a single PCR reporter using three successive reactions ? reverse transcription (RT) to convert viral RNA to cDNA, an oligonucleotide ligation assay (OLA) reaction to create a unique PCR target in the presence of drug resistance mutations, and PCR to amplify ligation products. Aim 1 optimizes a multiplex design for this assay. A major challenge with this RT-OLA-PCR approach is that the ligation reaction is incompatible with both the RT and PCR reaction conditions. To overcome incompatibility, in Aim 2, we propose to capitalize on our expertise using self-contained magnetic bead-based processing to maximize the limit of detection of the assay. The successful completion of this project will result in new drug resistance assays and benchtop processing methods that could be used to detect the major genotypic mutations in a setting with access to standard PCR. A subsequent R01 with our partners in Nigeria will incorporate these reagents and methods to validate with clinical samples the RT-OLA-PCR assay and sample processing appropriate for the workflow, clinic personnel, and existing infrastructure in Nigerian clinics providing antenatal care.